Electrolytic method of fuel

ABSTRACT

An electrolytic method of a fuel capable of suppressing reverse reaction of an enzyme and improving electrolytic rate is provided. In electrolyzing a fuel such as glucose by using an enzyme/electron mediator obtained by immobilizing an enzyme such as gluconate-5-dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase and an electron mediator onto a porous electrode made of a carbon material, electrode reaction is generated only in the enzyme/electron mediator electrode.

TECHNICAL FIELD

The present invention relates to an electrolytic method of a fuel in a fuel cell. More specifically, the present invention relates to a method of electrolyzing a fuel such as glucose by an electrode onto which an enzyme is immobilized in a bio-fuel cell.

BACKGROUND ART

A bio-fuel cell in which an enzyme as a catalyst is immobilized onto at least one of an anode and a cathode is able to efficiently extract electrons from a fuel such as glucose and ethanol incapable of being used with the use of a general industrial catalyst. Thus, the bio-fuel cell attracts attentions as a next generation fuel cell having high capacity and high safety (for example, see Patent documents 1 to 3).

FIG. 8 is a diagram illustrating reaction of an enzyme battery that includes a carbon electrode onto which an enzyme and an electron mediator are immobilized and that uses glucose as a fuel. In the enzyme battery illustrated in FIG. 8, oxidation reaction of glucose proceeds in an anode, and reduction reaction of oxygen (O₂) in the air proceeds in a cathode. In addition, in the anode, electrons are transferred in the order of glucose, glucose dehydrogenase, nicotinamide adenine dinucleotide (NAD+), diaphorase, the electron mediator, and the electrode (carbon).

CITATION LIST Patent Documents

-   Patent document 1: Japanese Unexamined Patent Application     Publication No. 2007-280944 -   Patent document 2: Japanese Unexamined Patent Application     Publication No. 2008-177088 -   Patent document 3: Japanese Unexamined Patent Application     Publication No. 2008-270206

SUMMARY OF THE INVENTION

However, the existing bio-fuel cell has a problem described below. That is, the enzymes used in the bio-fuel cell include an enzyme that initiates original reaction and reverse reaction concurrently. Thus, in the case where the enzyme initiating the reverse reaction is used, electrolytic time of the fuel is increased and power generation efficiency is lowered. Thus, in the past, an enzyme not initiating the reverse reaction has been developed by inheritable genetic modification or the like. If the enzyme not initiating the reverse reaction is used, electrolytic time of the fuel is able to be decreased and power generation efficiency is able to be improved. However, labor and cost are necessitated in order to realize practical use of such an enzyme.

Therefore, it is a main object of the present invention to provide an electrolytic method of a fuel capable of inhibiting reverse reaction of an enzyme and improving electrolytic rate.

In an electrolytic method of a fuel according to an embodiment of the present invention, electrolytic reaction is generated only in an electrode in electrolyzing the fuel by the electrode onto which an enzyme is immobilized.

In the present invention, since the fuel is electrolyzed in the electrode, reverse reaction of the enzyme is inhibited, and electrolytic rate is improved.

In the electrolytic method, an enzyme initiating reverse reaction is able to be used as the enzyme immobilized onto the electrode.

Further, an electron mediator may be immobilized onto the electrode together with the enzyme, and ratio between an oxidant and a reductant of the electron mediator may be controlled by changing electric potential applied between electrodes.

In this case, electric potential higher than half wave electric potential of the electron mediator is able to be applied between electrodes.

Furthermore, examples of the enzyme initiating reverse reaction include gluconate-5-dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase.

According to the embodiment of the present invention, since the fuel is electrolyzed in the electrode, enzyme reverse reaction is able to be inhibited, and electrolytic rate equal to or greater than that in the case of using an enzyme not initiating reverse reaction is able to be obtained without performing inheritable genetic modification or the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating reaction in which glucose is oxidized by an enzyme and four electrons are extracted.

FIG. 2 is a diagram schematically illustrating a method of in-electrode electrolysis according to Example of the present invention.

FIG. 3 is a diagram schematically illustrating a method of out-of-electrode electrolysis according to Comparative example.

FIGS. 4( a) and 4(b) are diagrams illustrating results of chronoamperometry performed by the method of Comparative example illustrated in FIG. 3. FIG. 4( a) illustrates a result of a case using a GDH immobilized electrode, and FIG. 4( b) illustrates a result of a case using a Gn5DH immobilized electrode.

FIGS. 5( a) and 5(b) are diagrams illustrating reaction rate of GHD. FIG. 5( a) illustrates forward reaction, and FIG. 5( b) illustrates reverse reaction.

FIGS. 6( a) and 6(b) are diagrams illustrating reaction rate of Gn5DH. FIG. 6( a) illustrates forward reaction, and FIG. 6( b) illustrates reverse reaction.

FIGS. 7( a) and 7(b) are diagrams illustrating results of chronoamperometry performed by the method of Example illustrated in FIG. 2. FIG. 7( a) illustrates a result of a case using the GDH immobilized electrode, and FIG. 7( b) illustrates a result of a case using the Gn5DH immobilized electrode.

FIG. 8 is a diagram illustrating reaction of an enzyme battery that includes a carbon electrode onto which an enzyme and an electron mediator are immobilized and that uses glucose as a fuel.

DESCRIPTION OF EMBODIMENTS

An embodiment of the present invention will be hereinafter described in detail with reference to the attached drawings. It is to be noted that the present invention is not limited to the embodiment described below.

[Whole Structure]

The inventors have keenly made experiments and examination in order to solve the problems in the existing bio-fuel cell. In the result, the inventors found the following fact. That is, in the case where electrolysis is made in a system in which a solution exists outside an electrode as in the bio-fuel cells described in Patent documents 1 to 3, difference between electrolytic time with the use of an enzyme initiating reverse reaction and electrolytic time with the use of an enzyme not initiating reverse reaction is particularly large.

FIG. 1 is a diagram illustrating reaction in which glucose is oxidized by an enzyme and four electrons are extracted. In this system, first, glucose is oxidized by glucose dehydrogenase (GDH) to obtain gluconolactone, and thereby two electrons are obtained with this reaction. Subsequently, the generated gluconolactone is degraded into 5-dehydrogluconate by gluconolactonase and gluconate-5-dehydrogenase (Gn5DH). In the case where an enzyme with high reverse reaction rate such as gluconate-5-dehydrogenase (Gn5DH) is used in such a system, electrolytic rate of the entire cell is decreased by rate limitation in the reaction contributed by the enzyme.

Thus, the inventors examined in-electrode electrolysis instead of the existing out-of-electrode electrolysis. In the result, the inventors found that electrolytic rate by the enzyme was improved in the in-electrode electrolysis, leading to the present invention. That is, in the electrolytic method of the present invention, in a bio-fuel cell including an electrode onto which an enzyme is immobilized, electrolytic reaction of a fuel is generated only in the electrode.

[Electrode]

In the electrolytic method of the present invention, the fuel is degraded by the enzyme immobilized onto the electrode to extract electrons, and protons (H⁺) is generated. As the electrode used at this time, an electrode made of a carbon material having internal voids and having a large surface area such as porous carbon, carbon pellet, carbon felt, and carbon paper is preferable. It is to be noted that the electrode material is not limited to the carbon material, and a metal material such as titanium, gold, copper, and nickel is able to be used.

[Enzyme]

The above-mentioned enzyme immobilized onto the electrode is able to be selected as appropriate according to the fuel used. For example, in the case where glucose is used as a fuel, glucose dehydrogenase (GDH) that oxidizes and degrades glucose is able to be used. Further, in the case where a monomeric sugar such as glucose is used, a coenzyme oxidase and an electron mediator are desirably immobilized together with an oxidase contributing to degradation of the fuel such as glucose dehydrogenase (GDH).

The coenzyme oxidase oxidizes a coenzyme reduced by the oxidase (for example, NAD+, NADP+ or the like) and a reductant of the coenzyme (for example, NADH, NADPH or the like). Examples thereof include diaphorase (DI). By action of the coenzyme oxidase, electrons are generated when the coenzyme is returned to the oxidant, and the electrons are transferred from the coenzyme oxidase to the electrode through the electron mediator.

Further, in the case where a polysaccharide is used as a fuel, in addition to the oxidase, the coenzyme oxidase, and the electron mediator, a degrading enzyme that encourages degradation such as hydrolysis of the polysaccharide to generate a monomeric sugar such as glucose is desirably immobilized. It is to be noted that in the specification, “polysaccharide” is polysaccharide in the broad sense of the term, means all carbohydrates from which a monomeric sugar with two or more molecules is generated by hydrolysis, and includes an oligosaccharide such as a disaccharide, a trisaccharide, and a tetrasaccharide. Specific examples of polysaccharide include starch, amylose, amylopectin, glycogen, cellulose, maltose, sucrose, and lactose. In such a polysaccharide, two or more monomeric sugars are bound. In any polysaccharide, glucose is contained as a monomeric sugar as a binding unit.

Moreover, amylose and amylopectin are components contained in starch. Starch is a mixture of amylose and amylopectin. For example, in the case where glucoamylase is used as a degradation enzyme of a polysaccharide, and glucose dehydrogenase (GDH) is used as an oxidase that degrades a monomeric sugar, a polysaccharide capable of being degraded to glucose by glucoamylase is able to be used. Specific examples of such a polysaccharide include starch, amylose, amylopectin, glycogen, and maltose. Here, Glucoamylase is a degradation enzyme that hydrolyzes α-glucan such as starch to generate glucose. Glucose dehydrogenase is an oxidase that oxidizes β-D-glucose to D-glucono-δ-lactone.

In addition, the electrolytic method of the present invention is in particular suitably applied to a system using an enzyme initiating reverse reaction such as gluconate-5-dehydrogenase (Gn5DH), alcohol dehydrogenase, and malate dehydrogenase.

[Electron Mediator]

As the electron mediator immobilized onto the electrode surface together with the foregoing enzyme, a compound having a quinone skeleton is preferably used, and specially a compound having a naphthoquinone skeleton is suitably used. Specific examples thereof include 2-amino-1,4-naphthoquinone (ANQ), 2-amino-3-methyl-1,4-naphthoquinone (AMNQ), 2-methyl-1,4-naphthoquinone (VK3), and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ). Further, as the compound having a quinone skeleton, in addition to the compound having a naphthoquinone skeleton, for example, anthraquinone and a derivative thereof are able to be used. Furthermore, according to needs, in addition to the compound having a quinone skeleton, one or more kinds of other compounds working as an electron mediator may be immobilized.

[Electrolytic Method]

In the electrolytic method of the present invention, electrolytic reaction is generated only in the electrode onto which an enzyme is immobilized. The method is not particularly limited, and examples thereof include a method of supplying an electrode surface with a fuel solution with a quantity corresponding to the surface area and a method of forming a micro flow path on the electrode surface and conducting a fuel solution through the flow path. Moreover, in this case, by monitoring the electrolytic amount and adjusting the fuel supply amount sequentially, significantly high electrolytic efficiency is able to be achieved. Further, other examples thereof may include a pump including feedback function in which a vaporized solution amount is calculated by using a mass sensor, and a vaporization portion of solution is refilled.

In electrolytic reaction, as electric potential applied between electrodes, electric potential higher than half wave potential of the electron mediator is desirably applied. Thereby, inside of the electrode is able to be in the environment with high oxidant/reductant ratio of the electron mediator, and entire reaction is able to be shifted in desirable direction.

As described above, in the electrolytic method of the present invention, electrolytic reaction is generated only in the electrode. Thus, dissolution of the electron mediator is able to be prevented. Further, ratio between the oxidant and the reductant of the electron mediator is able to be easily controlled by set electric potential. Thereby, it is possible to encourage reaction of the entire system to trend in the direction of original reaction, and to inhibit reverse reaction of the enzyme. Thus, the entire electrolytic rate is able to be improved. As a result, even if an enzyme initiating reverse reaction is used, power generation efficiency is able to be improved without performing enzyme modification or the like.

EXAMPLE

A description will be hereinafter given of effects of the present invention specifically with reference to Example and Comparative example of the present invention. FIG. 2 schematically illustrates a method of in-electrode electrolysis according to Example of the present invention. FIG. 3 schematically illustrates a method of out-of-electrode electrolysis according to Comparative example. In Example, the in-electrode electrolysis (Example) illustrated in FIG. 2 and the out-of-electrode electrolysis (Comparative example) illustrated in FIG. 3 were performed by using an electrode onto which gluconate-5-dehydrogenase (Gn5DH) was immobilized, and fuel electrolytic time was measured. Further, for comparison, similar measurement was performed for an electrode onto which glucose dehydrogenase (GDH) was immobilized.

In forming each electrode onto which each enzyme was immobilized, first, the following respective solutions (1) to (7) were prepared. In addition, as a buffer solution, 100 mM of sodium dihydrogen phosphate (NaH₂PO₄) buffer solution (I.S.: 0.3, pH: 7.0) was used. Moreover, the buffer solution in which each enzyme is dissolved is desirably kept in cold storage until just before usage. The prepared enzyme buffer solution is also desirably kept in cold storage as much as possible.

(1) DI Enzyme Buffer Solution

5 to 50 mg of diaphorase (DI) (EC1.6.99, manufactured by Unitika Ltd., B1D111) was weighed, and the resultant portion was dissolved in 1 ml of the buffer solution.

(2) GDH Enzyme Buffer Solution

5 to 50 mg of glucose dehydrogenase (GDH) (NAD-dependent, EC1.1.1.47, manufactured by Toyobo Co., Ltd., GLD-311) was weighed, and the resultant portion was dissolved in 1 ml of the buffer solution.

(3) Gn5DH Enzyme Buffer Solution

5 to 50 mg of gluconate-5-dehydrogenase (Gn5DH) (NAD-dependent, EC1.1.1.69, manufactured by Amano Enzyme Inc.) was weighed, and the resultant portion was dissolved in 1 ml of the buffer solution.

(4) NADH Buffer Solution

10 to 50 mg of NADH (manufactured by Sigma-Aldrich Corporation, N-8129) was weighed, and the resultant portion was dissolved in 0.1 ml of the buffer solution.

(5) ANQ Acetone Solution

10 to 50 mg of 2-amino-1,4-naphthoquinone (ANQ) (synthetic compound) was weighed, and the resultant portion was dissolved in 1 ml of acetone.

(6) PLL Aqueous Solution

An appropriate amount of poly-L-lysine hydrobromate (PLL) (manufactured by Wako Pure Chemical Industries, Ltd., 164-16961) was weighed, and the resultant portion was dissolved in ion-exchange water to obtain 2 wt % solution.

(7) PAAcNa Aqueous Solution

An appropriate amount of sodium polyacrylate (PAAcNa) (manufactured by Sigma-Aldrich Corporation, 041-00595) was weighed, and the resultant portion was dissolved in ion-exchange water to obtain 0.22 wt % solution.

Next, each given amount of the foregoing respective solutions was prepared and mixed to obtain a mixed solution. A porous carbon electrode was coated with the mixed solution, was subsequently dried, and an enzyme/electron-mediator-coated electrode was formed. Each blending quantity of each solution in the mixed solution with which the porous electrode was coated is shown in the following Table 1.

TABLE 1 Solution Blending quantity (1) DI enzyme buffer solution 50 μL (2) GDH enzyme buffer solution 50 μL or (3) Gn5DH enzyme buffer solution (4) NADH buffer solution 50 μL (5) ANQ acetone solution 50 μL

Next, the enzyme/electron-mediator-coated electrode was further coated with 50 μL of (6) PLL aqueous solution (corresponding to 0.2 μg of PLL) and 50 μL of (7) PAAcNa aqueous solution (corresponding to 0.003 μg of PAAcNa), is subsequently dried, and an enzyme/electron mediator immobilized electrode was formed. Out of the foregoing each enzyme/electron mediator immobilized electrode, an electrode coated with a mixed solution containing the GDH enzyme buffer solution will be hereinafter referred to as “GDH immobilized electrode,” and an electrode coated with a mixed solution containing the Gn5DH enzyme buffer solution will be hereinafter referred to as “Gn5DH immobilized electrode.”

Regarding an enzyme/electron mediator immobilized electrode 1 formed as above, 0.1 V as electric potential sufficiently higher than half wave electric potential of the electron mediator was set with respect to a reference electrode 2 (Ag|AgCl), and chronoamperometry was performed by the methods illustrated in FIG. 2 and FIG. 3. At this time, as a fuel solution, a solution obtained by dissolving glucose or gluconic acid as a fuel in 2 M imidazole buffer solution (pH: 7.0) so that the concentration became 0.4 M was used. These fuel solutions will be hereinafter referred to as “0.4 M glucose fuel solution” and “0.4 M gluconic acid fuel solution,” respectively.

Further, as Comparative example, out-of-electrode electrolysis of glucose or gluconic acid was performed by the electrochemical measurement method based on three-electrode method illustrated in FIG. 3. At this time, electrolysis was performed while 2 ml (0.8 mmol) of the 0.4 M glucose fuel solution or the 0.4 M gluconic acid fuel solution was thrown in and the solution was stirred by a stirrer 5. In the out-of-electrode electrolysis of Comparative example, the enzyme/electron mediator immobilized electrode 1 was used as an anode (work electrode), and a platinum wire 3 was used as a counter electrode.

FIGS. 4( a) and 4(b) are diagrams illustrating results of chronoamperometry performed by the method of Comparative example illustrated in FIG. 3. FIG. 4( a) illustrates a result of a case using the GDH immobilized electrode, and FIG. 4( b) illustrates a result of a case using the Gn5DH immobilized electrode. As illustrated in FIGS. 4( a) and 4(b), in the case where the out-of electrode electrolysis was performed by using the Gn5DH immobilized electrode, it took about 20 times as many as in the case of using the GDH immobilized electrode to complete all electrolysis. Thereby, it was found that the electrolysis rate of the Gn5DH immobilized electrode was significantly slow.

Therefore, oxygen activity measurement of respective forward reactions and reverse reactions in GDH and Gn5DH was performed by using ultraviolet light (UV). The detection wavelength was 340 nm, and the spectroscopic measurement cell having a light path length of 1 cm was used. As a measurement solution, a phosphate buffer solution (pH: 7.0) containing 10 mM of glucose or gluconic acid was used. Each NAD+ concentration of each measurement solution was adjusted to obtain the entire amount of 3 ml. Further, reaction was started by adding the enzyme to the prepared measurement solution. A rate at which NADH was generated from NAD+ (ΔABS/min) was regarded as reaction rate of each enzyme.

FIGS. 5( a) and 5(b) are diagrams illustrating reaction rate of GHD. FIG. 5( a) illustrates forward reaction, and FIG. 5( b) illustrates reverse reaction. FIGS. 6( a) and 6(b) are diagrams illustrating reaction rate of Gn5DH. FIG. 6( a) illustrates forward reaction, and FIG. 6( b) illustrates reverse reaction. As illustrated in FIGS. 5( a) and 5(b) and FIGS. 6( a) and 6(b), it was confirmed that reverse reaction rate of Gn5DH was significantly higher than that of GDH.

Therefore, next, as Example of the present invention, in-electrode electrolysis of glucose or gluconic acid was performed by the electrochemical measurement method based on three-electrode method illustrated in FIG. 2. At this time, electrolysis was performed by dropping 2 μL of the 0.4 M glucose fuel solution or the 0.4 M gluconic acid fuel solution onto the surface of the enzyme/electron mediator immobilized electrode 1 (the GDH immobilized electrode or the Gn5DH immobilized electrode). In the in-electrode electrolysis of Example, the enzyme/electron mediator immobilized electrode 1 was used as an anode (work electrode), a platinum mesh 6 was used as a counter electrode, and an insulator (paper) 7 was provided between the enzyme/electron mediator immobilized electrode 1 and the platinum mesh 6.

FIGS. 7( a) and 7(b) are diagrams illustrating results of chronoamperometry performed by the method of Example illustrated in FIG. 2. FIG. 7( a) illustrates a result of a case using the GDH immobilized electrode, and FIG. 7( b) illustrates a result of a case using the Gn5DH immobilized electrode. As illustrated in FIGS. 7( a) and 7(b), in the in-electrode electrolysis, both in the case of using the Gn5DH immobilized electrode and in the case of using the GDH immobilized electrode, electrolysis was completed within from 2000 to 3000 seconds both inclusive, showing almost no difference in electrolysis time.

From the foregoing results, it was confirmed that by applying the electrolytic method of the present invention, enzyme reverse reaction was able to be suppressed without performing inheritable genetic modification or the like. In addition, it was confirmed that by applying the electrolytic method of the present invention, even if an enzyme initiating reverse reaction was used, electrolytic rate equal to or greater than that in the case of using an enzyme not initiating reverse reaction was able to be obtained. 

1. An electrolytic method of a fuel in which electrolytic reaction is generated only in an electrode in electrolyzing the fuel by the electrode onto which an enzyme is immobilized.
 2. The electrolytic method of a fuel according to claim 1, wherein an enzyme initiating reverse reaction is used as the enzyme.
 3. The electrolytic method of a fuel according to claim 1, wherein an electron mediator is immobilized onto the electrode together with the enzyme, and ratio between an oxidant and a reductant of the electron mediator is controlled by changing electric potential applied between electrodes.
 4. The electrolytic method of a fuel according to claim 3, wherein electric potential higher than half wave electric potential of the electron mediator is applied.
 5. The electrolytic method of a fuel according to claim 2, wherein the enzyme is gluconate-5-dehydrogenase, alcohol dehydrogenase, or malate dehydrogenase. 